In tunnelling using tunnel boring machine (TBM), the surrounding rocks are typically supported by precast lining segments, pea-gravel backfilling and grouting. The compactness of the backfill layer is critical for ensuring the safety of the tunnel construction. However, poor fluidity of cement generally leads to uneven grouting effects, resulting in lower density in certain areas of the backfill layer. In contrast, enzyme-induced carbonate precipitation (EICP) is an environmentally friendly and sustainable technique which has superior mobility and diffusivity compared to cement. To investigate the reinforcement effects of EICP technique on backfill layer, a series of bio-cemented sand column tests and model tests were conducted in this study. The optimal working range of pea gravel and sand for effective bio-cementation were determined by comparing the permeability, unconfined compressive strength (UCS), calcium carbonate content (CCC), and wave velocity of bio-cemented sand columns. The effects and homogeneity of reinforcement based on model tests were assessed by point load tests, wave velocity measurements, and calcium carbonate content evaluations. The model tests with different grouting hole layout density were conducted to obtain the optimal hole placement scheme. The column test results demonstrated that the optimal working range of pea gravel to sand ratio for effective bio-cementation is 1.25–1.5. As the number of grouting cycles increases, the point load strength, wave velocity, CCC and UCS of the specimens increase while the permeability of the specimens decreases. The point load strength of bio-cemented specimens could reach up to 16.53 MPa, while the permeability was reduced by three orders of magnitude compared with that of untreated specimens. The EICP has been demonstrated to be an effective technique capable of improving the compactness and strength of the backfill layer, with aggregates effectively cemented by calcium carbonate generated. The model test results demonstrated that the full-coverage arrangement hole scheme achieves uniform cementation, while the space arrangement hole scheme produces concentrated CaCO3 near grouting holes. Furthermore, a new and improved grouting scheme is proposed based on the model test results. The data obtained in this study offer valuable references for the reinforcement of the backfill layer in TBM tunnelling using the EICP technology.